The influenza A virus genome consists of eight negative-sense single-stranded RNA segments. Synthesis of viral mRNA is catalyzed by a viral-encoded RNA-dependent RNA polymerase (E.C. 2.7.7.48). Influenza virus transcription is initiated by a mechanism in which primers are generated by cleaving host cell transcripts 10-13 nucleotides from their 5' cap structure. The cleavage and priming reactions are dependent on the transcript possessing a cap-1 structure containing a 7-methylated terminal G and a 2'-O-methylated penultimate purine base (7mGpppRm). The influenza endonuclease represents an attractive target for development of antiviral agents, and recently both small molecule (Tomassini et al, 1994 AntiMicrob. Agents and Chemo. 38:2827-2837) and oligonucleotide inhibitors (Chung et al 1994 Proc. Natl. Acad. Sci. USA 91:2372-2376) have been described.
Screening for potential inhibitors of the influenza endonuclease has been hampered, however, by the lack of a suitable assay method. An ideal assay system should have: a) high throughput; b) the ability to distinguish influenza endonuclease-catalyzed cleavage from nonspecific RNA cleavage; and c) high sensitivity. Previous endonuclease assays involved the use of polyacrylamide gel electrophoresis to separate product from substrate (Plotch et al, 1981 Cell 23: 847-858) which is not convenient for processing large numbers of samples. Assays have also been described for the overall influenza transcriptase reaction which may be capable of detecting inhibition of the endonuclease (Plotch et al, 1977 J. Virol. 21:24-34.). However, the overall reaction is a complex, multistep process in which cleavage may not be rate-limiting and endonuclease inhibitors may be missed.